Phosphoenolpyruvate carboxykinase (, PEPCK) is an

enzyme An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...

in the

lyase In biochemistry, a lyase is an enzyme that catalyzes the breaking (an elimination reaction) of various chemical bonds by means other than hydrolysis (a substitution reaction) and oxidation Redox ( , , reduction–oxidation or oxidatio ...

family used in the metabolic pathway of

gluconeogenesis Gluconeogenesis (GNG) is a metabolic pathway that results in the biosynthesis of glucose from certain non-carbohydrate carbon substrates. It is a ubiquitous process, present in plants, animals, fungi, bacteria, and other microorganisms. In verte ...

. It converts

oxaloacetate Oxaloacetic acid (also known as oxalacetic acid or OAA) is a crystalline organic compound with the chemical formula HO2CC(O)CH2CO2H. Oxaloacetic acid, in the form of its conjugate base oxaloacetate, is a metabolic intermediate in many processes ...

into

phosphoenolpyruvate Phosphoenolpyruvate (2-phosphoenolpyruvate, PEP) is the carboxylic acid derived from the enol of pyruvate and a phosphate anion. It exists as an anion. PEP is an important intermediate in biochemistry. It has the high-energy phosphate, highest-e ...

and

carbon dioxide Carbon dioxide is a chemical compound with the chemical formula . It is made up of molecules that each have one carbon atom covalent bond, covalently double bonded to two oxygen atoms. It is found in a gas state at room temperature and at norma ...

. It is found in two forms,

cytosol The cytosol, also known as cytoplasmic matrix or groundplasm, is one of the liquids found inside cells ( intracellular fluid (ICF)). It is separated into compartments by membranes. For example, the mitochondrial matrix separates the mitochondri ...

ic and

mitochondrial A mitochondrion () is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is used ...

Structure

In humans there are two isoforms of PEPCK; a cytosolic form (SwissProt P35558) and a mitochondrial isoform (SwissProt Q16822) which have 63.4% sequence identity. The cytosolic form is important in gluconeogenesis. However, there is a known transport mechanism to move PEP from the mitochondria to the cytosol, using specific membrane transport proteins. PEP transport across the inner mitochondrial membrane involves the mitochondrial tricarboxylate transport protein and to a lesser extent the adenine nucleotide carrier. The possibility of a PEP/pyruvate transporter has also been put forward. X-ray structures of PEPCK provide insight into the structure and the mechanism of PEPCK enzymatic activity. The mitochondrial isoform of chicken liver PEPCK complexed with Mn²⁺, Mn2+-

(PEP), and Mn²⁺-GDP provides information about its structure and how this enzyme catalyzes reactions. Delbaere et al. (2004) resolved PEPCK in ''E. coli'' and found the

active site In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...

sitting between a

C-terminal domain The C-terminus (also known as the carboxyl-terminus, carboxy-terminus, C-terminal tail, carboxy tail, C-terminal end, or COOH-terminus) is the end of an amino acid chain (protein or polypeptide), terminated by a free carboxyl group (-COOH). When t ...

and an

N-terminal domain The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...

. The active site was observed to be closed upon rotation of these domains. Phosphoryl groups are transferred during PEPCK action, which is likely facilitated by the

eclipsed conformation In chemistry an eclipsed conformation is a conformation in which two substituents X and Y on adjacent atoms A, B are in closest proximity, implying that the torsion angle X–A–B–Y is 0°. Such a conformation can exist in any open chain, ...

of the phosphoryl groups when ATP is bound to PEPCK. Since the eclipsed formation is one that is high in energy, phosphoryl group transfer has a decreased

energy of activation In the Arrhenius model of reaction rates, activation energy is the minimum amount of energy that must be available to reactants for a chemical reaction to occur. The activation energy (''E''a) of a reaction is measured in kilojoules per mole (k ...

, meaning that the groups will transfer more readily. This transfer likely happens via a mechanism similar to

SN2 The bimolecular nucleophilic substitution (SN2) is a type of reaction mechanism that is common in organic chemistry. In the SN2 reaction, a strong nucleophile forms a new bond to an sp3-hybridised carbon atom via a backside attack, all while t ...

displacement.

In different species

PEPCK gene

transcription Transcription refers to the process of converting sounds (voice, music etc.) into letters or musical notes, or producing a copy of something in another medium, including: Genetics * Transcription (biology), the copying of DNA into RNA, often th ...

occurs in many species, and the amino acid sequence of PEPCK is distinct for each species. For example, its structure and its specificity differ in humans, ''Escherichia coli'' (''

E. coli ''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus ''Escherichia'' that is commonly foun ...

''), and the parasite''

Trypanosoma cruzi ''Trypanosoma cruzi'' is a species of parasitic euglenoids. Among the protozoa, the trypanosomes characteristically bore tissue in another organism and feed on blood (primarily) and also lymph. This behaviour causes disease or the likelihood ...

''.

Mechanism

PEPCKase converts

into

and

. Image:Oxaloacetic acid.svg,

Image:Phosphoenolpyruvic acid.svg,

As PEPCK acts at the junction between

glycolysis Glycolysis is the metabolic pathway that converts glucose () into pyruvic acid, pyruvate and, in most organisms, occurs in the liquid part of cells (the cytosol). The Thermodynamic free energy, free energy released in this process is used to form ...

and the Krebs cycle, it causes

decarboxylation Decarboxylation is a chemical reaction that removes a carboxyl group and releases carbon dioxide (CO2). Usually, decarboxylation refers to a reaction of carboxylic acids, removing a carbon atom from a carbon chain. The reverse process, which is ...

of a C₄ molecule, creating a C₃ molecule. As the first committed step in gluconeogenesis, PEPCK decarboxylates and

phosphorylate In biochemistry, phosphorylation is described as the "transfer of a phosphate group" from a donor to an acceptor. A common phosphorylating agent (phosphate donor) is ATP and a common family of acceptor are alcohols: : This equation can be writt ...

(OAA) for its conversion to PEP, when GTP is present. As a phosphate is transferred, the reaction results in a GDP molecule. When

pyruvate kinase Pyruvate kinase is the enzyme involved in the last step of glycolysis. It catalyzes the transfer of a phosphate group from phosphoenolpyruvate (PEP) to adenosine diphosphate (ADP), yielding one molecule of pyruvate and one molecule of ATP. Pyruv ...

– the enzyme that normally catalyzes the reaction that converts PEP to pyruvate – is knocked out in mutants of ''

Bacillus subtilis ''Bacillus subtilis'' (), known also as the hay bacillus or grass bacillus, is a gram-positive, catalase-positive bacterium, found in soil and the gastrointestinal tract of ruminants, humans and marine sponges. As a member of the genus ''Bacill ...

'', PEPCK participates in one of the replacement

anaplerotic reactions Anaplerotic reactions, a term coined by Hans Kornberg and originating from the Greeἀνά 'up' anπληρόω 'to fill', are chemical reactions that form intermediates of a metabolic pathway. Examples of such are found in the citric acid cycle (TC ...

, working in the reverse direction of its normal function, converting PEP to OAA. Although this reaction is possible, the kinetics are so unfavorable that the mutants grow at a very slow pace or do not grow at all.

Function

Gluconeogenesis

PEPCK-C catalyzes an irreversible step of

, the process whereby glucose is synthesized. The enzyme has therefore been thought to be essential in glucose homeostasis, as evidenced by laboratory mice that contracted

diabetes mellitus type 2 Type 2 diabetes (T2D), formerly known as adult-onset diabetes, is a form of diabetes mellitus that is characterized by high blood sugar, insulin resistance, and relative lack of insulin. Common symptoms include increased thirst, frequent ...

as a result of the overexpression of PEPCK-C.Vanderbilt Medical Center. "Granner Lab, PEPCK Research." 2001. Online. Internet. Accessed 10:46PM, 4/13/07. www.mc.vanderbilt.edu/root/vumc.php?site=granner&doc=119 The role that PEPCK-C plays in gluconeogenesis may be mediated by the

citric acid cycle The citric acid cycle—also known as the Krebs cycle, Szent–Györgyi–Krebs cycle, or TCA cycle (tricarboxylic acid cycle)—is a series of chemical reaction, biochemical reactions that release the energy stored in nutrients through acetyl-Co ...

, the activity of which was found to be directly related to PEPCK-C abundance. PEPCK-C levels alone were not highly correlated with gluconeogenesis in the mouse liver, as previous studies have suggested. While the mouse liver almost exclusively expresses PEPCK-C, humans equally present a mitochondrial isozyme (PEPCK-M). PEPCK-M has gluconeogenic potential per se. Therefore, the role of PEPCK-C and PEPCK-M in gluconeogenesis may be more complex and involve more factors than was previously believed.

Animals

In animals, this is a rate-controlling step of

, the process by which cells synthesize

glucose Glucose is a sugar with the Chemical formula#Molecular formula, molecular formula , which is often abbreviated as Glc. It is overall the most abundant monosaccharide, a subcategory of carbohydrates. It is mainly made by plants and most algae d ...

from metabolic precursors. The blood glucose level is maintained within well-defined limits in part due to precise regulation of PEPCK gene expression. To emphasize the importance of PEPCK in glucose

homeostasis In biology, homeostasis (British English, British also homoeostasis; ) is the state of steady internal physics, physical and chemistry, chemical conditions maintained by organism, living systems. This is the condition of optimal functioning fo ...

, over expression of this enzyme in mice results in symptoms of type II

diabetes mellitus Diabetes mellitus, commonly known as diabetes, is a group of common endocrine diseases characterized by sustained hyperglycemia, high blood sugar levels. Diabetes is due to either the pancreas not producing enough of the hormone insulin, or th ...

, by far the most common form of diabetes in humans. Due to the importance of blood glucose homeostasis, a number of

hormones A hormone (from the Greek participle , "setting in motion") is a class of signaling molecules in multicellular organisms that are sent to distant organs or tissues by complex biological processes to regulate physiology and behavior. Hormones a ...

regulate a set of

genes In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...

(including PEPCK) in the

liver The liver is a major metabolic organ (anatomy), organ exclusively found in vertebrates, which performs many essential biological Function (biology), functions such as detoxification of the organism, and the Protein biosynthesis, synthesis of var ...

that modulate the rate of glucose synthesis. PEPCK-C is controlled by two different hormonal mechanisms. PEPCK-C activity is increased upon the secretion of both

cortisol Cortisol is a steroid hormone in the glucocorticoid class of hormones and a stress hormone. When used as medication, it is known as hydrocortisone. Cortisol is produced in many animals, mainly by the ''zona fasciculata'' of the adrenal corte ...

from the adrenal cortex and

glucagon Glucagon is a peptide hormone, produced by alpha cells of the pancreas. It raises the concentration of glucose and fatty acids in the bloodstream and is considered to be the main catabolic hormone of the body. It is also used as a Glucagon (medic ...

from the alpha cells of the pancreas. Glucagon indirectly elevates the expression of PEPCK-C by increasing the levels of cAMP (via activation of adenylyl cyclase) in the liver which consequently leads to the phosphorylation of S133 on a beta sheet in the

CREB CREB-TF (CREB, cAMP response element-binding protein) is a cellular transcription factor. It binds to certain DNA sequences called cAMP response elements (CRE), thereby increasing or decreasing the transcription of the genes. CREB was first des ...

protein. CREB then binds upstream of the PEPCK-C gene at CRE (cAMP response element) and induces PEPCK-C transcription. Cortisol on the other hand, when released by the adrenal cortex, passes through the lipid membrane of liver cells (due to its hydrophobic nature it can pass directly through cell membranes) and then binds to a Glucocorticoid Receptor (GR). This receptor dimerizes and the cortisol/GR complex passes into the nucleus where it then binds to the Glucocorticoid Response Element (GRE) region in a similar manner to CREB and produces similar results (synthesis of more PEPCK-C). Together, cortisol and glucagon can have huge synergistic results, activating the PEPCK-C gene to levels that neither cortisol or glucagon could reach on their own. PEPCK-C is most abundant in the liver, kidney, and adipose tissue. A collaborative study between the U.S. Environmental Protection Agency (EPA) and the University of New Hampshire investigated the effect of DE-71, a commercial

PBDE Polybrominated diphenyl ethers or PBDEs, are a class of organobromine compounds that are used as flame retardants. Like other brominated flame retardants, PBDEs have been used in a wide array of products, including building materials, electronic ...

mixture, on PEPCK enzyme kinetics and determined that in vivo treatment of the environmental pollutant compromises liver glucose and lipid metabolism possibly by activation of the pregnane xenobiotic receptor (

PXR In the field of molecular biology, the pregnane X receptor (PXR), also known as the steroid and xenobiotic sensing nuclear receptor (SXR) or nuclear receptor subfamily 1, group I, member 2 (NR1I2) is a protein that in humans is encoded by the ' ...

), and may influence whole-body insulin sensitivity. Researchers at Case Western Reserve University have discovered that overexpression of cytosolic PEPCK in skeletal muscle of mice causes them to be more active, more aggressive, and have longer lives than normal mice; see '' metabolic supermice''.

Plants

PEPCK () is one of three decarboxylation enzymes used in the inorganic carbon concentrating mechanisms of C₄ and

CAM Cam or CAM may refer to: Science and technology * Cam (mechanism), a mechanical linkage which translates motion * Camshaft, a shaft with a cam * Camera or webcam, a device that records images or video In computing * Computer-aided manufacturin ...

plants. The others are NADP-malic enzyme and NAD-malic enzyme. In C₄ carbon fixation,

is first fixed by combination with

to form

in the

mesophyll A leaf (: leaves) is a principal appendage of the stem of a vascular plant, usually borne laterally above ground and specialized for photosynthesis. Leaves are collectively called foliage, as in "autumn foliage", while the leaves, stem, f ...

. In PEPCK-type C₄ plants the

is then converted to

aspartate Aspartic acid (symbol Asp or D; the ionic form is known as aspartate), is an α-amino acid that is used in the biosynthesis of proteins. The L-isomer of aspartic acid is one of the 22 proteinogenic amino acids, i.e., the building blocks of protein ...

, which travels to the

bundle sheath A vascular bundle is a part of the transport system in vascular plants. The transport itself happens in the Plant stem, stem, which exists in two forms: xylem and phloem. Both these tissues are present in a vascular bundle, which in addition ...

. In the

cells Cell most often refers to: * Cell (biology), the functional basic unit of life * Cellphone, a phone connected to a cellular network * Clandestine cell, a penetration-resistant form of a secret or outlawed organization * Electrochemical cell, a d ...

is converted back to

. PEPCK decarboxylates the

, releasing

, which is then fixed by the enzyme

Rubisco Ribulose-1,5-bisphosphate carboxylase/oxygenase, commonly known by the abbreviations RuBisCo, rubisco, RuBPCase, or RuBPco, is an enzyme () involved in the light-independent (or "dark") part of photosynthesis, including the carbon fixation by wh ...

. For each molecule of carbon dioxide produced by PEPCK, a molecule of ATP is consumed. PEPCK acts in plants that undergo C₄ carbon fixation, where its action has been localized to the

, in contrast to mammals, where it has been found that PEPCK works in

mitochondria A mitochondrion () is an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and use aerobic respiration to generate adenosine triphosphate (ATP), which is us ...

. Although it is found in many different parts of plants, it has been seen only in specific cell types, including the areas of the

phloem Phloem (, ) is the living tissue in vascular plants that transports the soluble organic compounds made during photosynthesis and known as ''photosynthates'', in particular the sugar sucrose, to the rest of the plant. This transport process is ...

. It has also been discovered that, in cucumber (''Cucumis sativus L''.), PEPCK levels are increased by multiple effects that are known to decrease the cellular pH of plants, although these effects are specific to the part of the plant. PEPCK levels rose in roots and stems when the plants were watered with

ammonium chloride Ammonium chloride is an inorganic chemical compound with the chemical formula , also written as . It is an ammonium salt of hydrogen chloride. It consists of ammonium cations and chloride anions . It is a white crystalline salt (chemistry), sal ...

at a low pH (but not at high pH), or with

butyric acid Butyric acid (; from , meaning "butter"), also known under the systematic name butanoic acid, is a straight-chain alkyl carboxylic acid with the chemical formula . It is an oily, colorless liquid with an unpleasant odor. Isobutyric acid (2-met ...

. However, PEPCK levels did not increase in leaves under these conditions. In leaves, 5% content in the atmosphere leads to higher PEPCK abundance.

Bacteria

In an effort to explore the role of PEPCK, researchers caused the overexpression of PEPCK in ''E. coli'' bacteria via

recombinant DNA Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fo ...

. PEPCK of ''

Mycobacterium tuberculosis ''Mycobacterium tuberculosis'' (M. tb), also known as Koch's bacillus, is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, ''M. tuberculosis'' ha ...

'' has been shown to trigger the immune system in mice by increasing

cytokine Cytokines () are a broad and loose category of small proteins (~5–25 kDa) important in cell signaling. Cytokines are produced by a broad range of cells, including immune cells like macrophages, B cell, B lymphocytes, T cell, T lymphocytes ...

activity. As a result, it has been found that PEPCK may be an appropriate ingredient in the development of an effective subunit vaccination for

tuberculosis Tuberculosis (TB), also known colloquially as the "white death", or historically as consumption, is a contagious disease usually caused by ''Mycobacterium tuberculosis'' (MTB) bacteria. Tuberculosis generally affects the lungs, but it can al ...

Clinical significance

Activity in cancer

PEPCK has not been considered in cancer research until recently. It has been shown that in human tumor samples and human cancer cell lines (breast, colon and lung cancer cells) PEPCK-M, and not PEPCK-C, was expressed at enough levels to play a relevant metabolic role. Therefore, PEPCK-M could have a role in cancer cells, especially under nutrient limitation or other stress conditions.

Regulation

In humans

PEPCK-C is enhanced, both in terms of its production and activation, by many factors. Transcription of the PEPCK-C gene is stimulated by

glucocorticoids Glucocorticoids (or, less commonly, glucocorticosteroids) are a class of corticosteroids, which are a class of steroid hormones. Glucocorticoids are corticosteroids that bind to the glucocorticoid receptor that is present in almost every vertebra ...

retinoic acid Retinoic acid (simplified nomenclature for all-''trans''-retinoic acid) is a metabolite of vitamin A1 (all-''trans''-retinol) that is required for embryonic development, male fertility, regulation of bone growth and immune function. All-''trans ...

, and adenosine 3',5'-monophosphate (

cAMP Camp may refer to: Areas of confinement, imprisonment, or for execution * Concentration camp, an internment camp for political prisoners or politically targeted demographics, such as members of national or minority ethnic groups * Extermination ...

), while it is inhibited by

insulin Insulin (, from Latin ''insula'', 'island') is a peptide hormone produced by beta cells of the pancreatic islets encoded in humans by the insulin (''INS)'' gene. It is the main Anabolism, anabolic hormone of the body. It regulates the metabol ...

. Of these factors, insulin, a hormone that is deficient in the case of type 1 diabetes mellitus, is considered dominant, as it inhibits the transcription of many of the stimulatory elements. PEPCK activity is also inhibited by hydrazine sulfate, and the inhibition therefore decreases the rate of gluconeogenesis. In prolonged

acidosis Acidosis is a biological process producing hydrogen ions and increasing their concentration in blood or body fluids. pH is the negative log of hydrogen ion concentration and so it is decreased by a process of acidosis. Acidemia The term ac ...

, PEPCK-C is upregulated in

renal proximal tubule brush border cell The proximal tubule is the segment of the nephron in kidneys which begins from the renal (tubular) pole of the Bowman's capsule to the beginning of loop of Henle. At this location, the glomerular parietal epithelial cells (PECs) lining bowman’s ...

s, in order to secrete more NH₃ and thus to produce more HCO₃⁻. The GTP-specific activity of PEPCK is highest when Mn²⁺ and Mg²⁺ are available. In addition, hyper-reactive

cysteine Cysteine (; symbol Cys or C) is a semiessential proteinogenic amino acid with the chemical formula, formula . The thiol side chain in cysteine enables the formation of Disulfide, disulfide bonds, and often participates in enzymatic reactions as ...

(C307) is involved in the binding of Mn²⁺ to the active site.

Plants

As discussed previously, PEPCK abundance increased when plants were watered with low-pH ammonium chloride, though high pH did not have this effect.

Classification

It is classified under EC number 4.1.1. There are three main types, distinguished by the source of the energy to drive the reaction: * 4.1.1.32 – GTP (

PCK1 Phosphoenolpyruvate carboxykinase 1 (soluble), also known as PCK1, is an enzyme which in humans is encoded by the ''PCK1'' gene. Function This enzyme is a main control point for the regulation of gluconeogenesis. The cytosolic enzyme encoded by ...

PCK2 Phosphoenolpyruvate carboxykinase 2, mitochondrial (PCK2, PEPCK-M), is an isozyme of phosphoenolpyruvate carboxykinase (PCK, PEPCK) that in humans is encoded by the ''PCK2'' gene on chromosome 14. This gene encodes a mitochondrial enzyme that cat ...

) * 4.1.1.38 –

diphosphate In chemistry, pyrophosphates are phosphorus oxyanions that contain two phosphorus atoms in a linkage. A number of pyrophosphate salts exist, such as disodium pyrophosphate () and tetrasodium pyrophosphate (), among others. Often pyrophosphate ...

* 4.1.1.49 – ATP

References

External links

* * * "mighty mice" (PEPCK-Cmus mice) https://web.archive.org/web/20071107175951/http://blog.case.edu/case-news/2007/11/02/mightymouse {{Portal bar, Biology, border=no EC 4.1.1